Aspirin and its Activities
A proper balance between the activities of coagulation and fibrinolytic cascades is needed both to protect an organism from excessive blood loss upon injury and to maintain blood flow within the vascular system. The two opposing coagulation and fibrinolytic cascades are recognized to comprise a series of zymogen to enzyme conversions which terminate in the two respective proteolytic enzymes, thrombin and plasmin. These enzymes catalyze the formation and removal of fibrin within the circulatory system. Imbalances are characterized by either bleeding or thrombotic tendencies which may result in heart attacks or strokes in the organism.
The anti-thrombotic effect of aspirin has long been recognized, and low doses of aspirin (acetylsalicylic acid; ASA) are recommended for the prevention of ischemic events in patients with coronary artery disease. In large clinical studies, aspirin has been shown to significantly reduce both the occurrence of myocardial infarction and mortality rate in patients with unstable angina pectoris that often occurs before a heart attack and/or previous myocardial infarction. Simoons, M., Lancet, 2001, 357(9272):1915–24.
The action of aspirin as well as other non-steroidal anti-inflammatory drugs (NSAIDS) is thought to derive mostly from the selective inhibition of cyclooxygenases 1 and 2(COX-1 and -2). At the platelet-vessel wall, aspirin at low doses selectively and irreversibly inhibits COX-1, which synthesizes thromboxane A2 (Tx-A2) and causes platelet activation. Hence, aspirin prevents blood platelets from aggregating, one of the initial steps in the formation of blood clots.
Aspirin is also a currently available therapy for several other diseases and disorders including fever, colon cancer, pancreatic cancer and inflammatory diseases such as arthritis. Aspirin is often a first-line therapy for rheumatoid arthritis, an inflammatory process which causes erosion or destruction of bone and cartilage joints. In addition to aspirin, current arthritis treatment regimens often employ ibuprofen and COX-2 inhibitors such as CELEBREX® (celecoxib) and VIOXX® (rofecoxib) which also act by reducing inflammation of the joints. Although these treatments are well established for arthritis sufferers, they often have unwanted or adverse side effects.
Thus, while aspirin and other drugs are often used for their known analgesic activity, antipyretic activity, anti-inflammatory activity, anti-platelet aggregation activity and prophylactic properties, aspirin's mechanism of action, and concomitantly, the mechanisms that lead to the production of unwanted side effects, are not yet fully understood. For example, the anti-platelet activity of aspirin cannot account for all the biological effects associated with aspirin therapy. Aspirin has been shown to reduce thrombin generation at the site of injury and can effect tissue factor-initiated coagulation. Undas et al., 2001, Blood, 98:2423–2431. Other studies suggest that aspirin therapy increases the fibrinolytic activity of plasma. Green, D., 2001, Clin. Cornerstone, 3:50–60. Further studies have been unable to attribute the mechanism by which aspirin enhances fibrinolysis to changes in tissue plasminogen activator levels. Bjornsson et al., 1989, J. Pharmacol. Exp. Ther., 250:154–161. Thus, there is confusion in the art regarding the mechanism of aspirin action, particularly in causing adverse side effects.
Thrombin Activatable Fibrinolysis Inhibitor
Thrombin activatable fibrinolysis inhibitor (“TAFI”) is a 60 kDa glycoprotein present in human plasma that modulates fibrinolysis in vivo. TAFI present in plasma is a proenzyme form which is most efficiently activated by proteolytic cleavage at Arg-92 with a thrombin-thrombomodulin complex. TAFI may also be activated by proteolytic cleavage by other proteolytic enzymes including, but not limited to, thrombin or plasmin (“activation of TAFI”). Upon activation of TAFI by proteolytic cleavage with thrombin-thrombomodulin, an active enzyme of 35 kDa is formed with carboxypeptidase-like activity (“TAFIa”). This molecule has also been referred to in the literature as plasma carboxypeptidase B (“PCPB”), or plasma carboxypeptidase U (“PCPU”). Tan et al., Biochemistry, 1995, 34:5811–5816; Wang et al., J. Biol. Chem. 269:15937 (1994); Nesheim et al., 1995, J. Biol. Chem. 270:14477.
Modulation of fibrinolysis occurs when TAFIa cleaves C-terminal arginine and lysine residues of partially degraded fibrin, thereby inhibiting the stimulation of tissue plasminogen activator (t-PA) modulated plasminogen activation. The fibrinolytic system is activated primarily by t-PA which is provided by damaged cells in the blood vessel wall. t-PA converts circulating plasminogen to the active protease plasmin and can produce either slow enhancement of fibrinolysis or, when combined with fibrin, rapid enhancement of fibrinolysis. The effect of t-PA on fibrinolysis can be blocked by a class of inhibitors termed plasminogen activator inhibitors (PAIs), of which several have been identified.
Thrombomodulin is a component of the blood vessel wall which binds thrombin and changes its specificity from fibrinogen to protein C, resulting in a molecule possessing anticoagulant, rather than procoagulant, activity. The thrombin-thrombomodulin complex catalyzes cleavage of protein C to activated protein C, which results in down-regulation of the coagulation cascade by proteolytically inactivating the essential cofactors, Factor Va and VIIIa. In this manner, the body regulates coagulation cascade.
Studies such as that by Taylor et al., 1985, Thromb. Res. 37:639 have suggested that activated protein C is not only an anticoagulant, but also profibrinolytic, both in vivo and in vitro. Subsequently, it was determined that protein C only appears profibrinolytic because it prevents the thrombin-catalyzed activation of a previously unknown fibrinolysis inhibitor, whose precursor was isolated from plasma and designated as being TAFI.
TAFI was discovered independently in three different laboratories. It initially appeared as an unstable carboxypeptidase B-like molecule in human serum and was described by Hendriks et al., 1990, Biochim. Biophys. Acta 1034:86. A year later the cDNA for the molecule was cloned, its amino acid sequence was described, its activation by trypsin and its enzymatic properties toward synthetic carboxypeptidase B substrates was reported (see U.S. Pat. No. 5,206,161). In 1994, Wang et al., (1994, J. Biol. Chem. 269:15937) isolated the activated molecule and named it carboxypeptidase U (“U” being designated for unstable). Subsequently, Nesheim et al. (1995, J. Biol. Chem. 270:14477) showed that the protein was both activated by thrombin and inhibits fibrinolysis, and designated the molecule TAFI. The co-identity of PCPB, PCPU, and TAFI has been established by their independent chromatographic behavior on plasminogen Sepharose® and the amino acid sequences present at the activation cleavage site.
The mechanism of TAFI inhibition of fibrinolysis can be schematically described as depicted in FIG. 5.
As TAFIa is believed to play a central regulatory role in the fibrinolytic cascade, the manipulation of TAFIa levels or activity in biological fluids has important therapeutic applications with respect to hemorrhagic disorders including, but not limited to, vascular and heart pathologies, and stroke. Inhibitors of TAFIa enhance fibrinolysis and have an anti-coagulant effect (see U.S. Pat. No. 5,993,815). Inhibitors of TAFIa could also be effective at treating or preventing the inflammation associated with arthritis as vascular endothelial growth factor (VEGF) is a potential substrate of TAFIa. VEGF has been linked with arthritis (Farva, R. A., 1994, J. Exp. Med. 180:341–6).
There remains a need in the art for methods of modulating TAFIa activity for therapeutic use in the treatment, prevention or management of hemorrhagic or thrombotic diseases or disorders. Moreover, there is a need to identify compounds that have the analgesic, antipyretic activity, anti-inflammatory activity, anti-platelet aggregation activity and prophylactic activity of aspirin without the hemorrhagic side effects. In addition there remains a need in the art for effective methods of screening compounds for TAFIa modulating activity which may be used in the treatment of fibrinolytic or thrombotic diseases or disorders.